MMP -7 is one of matrix metalloproteinases (hereinafter also referred to as “MMP”) belonging to a zinc metalloproteinase family which possesses a zinc molecule at the active site (cf. for instance, Non-patent reference 1). MMP is produced as a precursor, its signal sequence is processed upon extracellular secretion, and then a pro-sequence is processed to yield an active form. MMP extracellularly secreted controls metabolism of extracellular matrix. On the other hand, it is reported that MMP-7 is mainly secreted from cancer cells and is involved in invasion and metastasis (cf. for instance, Non-patent reference 2). MMP-7 lacks the hinge region and the hemopexin-like domain common in many of the other MMPs and consists of the minimum molecular unit as compared to the other MMPs. A substrate of MMP-7 is components constituting collagen or extracellular matrix (fibronectin, vitronectin, laminin, aggrecan).
MMP-7 is presumed to be involved in spontaneous remission of nucleus pulposus existing in the (spinal) epidural space viewing that a substrate of MMP-7 is aggrecan which is a main component of the cartilage tissue and that macrophages from specimen of intervertebral disk displacement surgery express MMP-7 (cf. for instance, Non-patent reference 3). Thereafter, Haro et al. administered MMP-7 to the intervertebral disk of hernial dog and observed the decrease in a volume of nucleus pulposus within the intervertebral disk to thereby show the possibility of MMP-7 as a medicament for treating intervertebral disk displacement (cf. for instance, Non-patent reference 4). Development of MMP-7 as a medicine is desired. However, MMP-7 occurs in the living body only in a trace amount and thus it is extremely difficult to isolate and purify MMP-7 from the living body. Besides, when components from the living body are used, there is a concern in view of safety such as potential viral infection. Although MMP-7 can be obtained from cancer cells, it is not preferable to use cancer cells as a source for production (cf. for instance, Non-patent reference 5).
For dissolving such problems, an attempt has been made to obtain MMP-7 by a recombinant DNA technology. There are the report by Barnett et al. that MMP-7 is expressed in CHO cells (cf. for instance, Non-patent reference 6), the report that, by using a nucleic acid fragment generated by linking a nucleotide sequence of a signal sequence of alkaline phosphatase to a gene sequence of pro-matrix metalloproteinase 7 (hereinafter also referred to as “proMMP-7”) optimized for codon usage of E. coli, soluble proMMP-7 was expressed at 34° C. and insoluble proMMP-7 was expressed at 42° C. (cf. for instance, Patent reference 1) and the report that, by using a nucleic acid fragment generated by linking a modified signal peptide to a gene fragment of proMMP-7, proMMP-7 is expressed as an inclusion body in a large amount (cf. for instance, Patent reference 2).
For conversion of proMMP-7 to active MMP-7, it is reported that proMMP-7 is heated at 37° C. in the presence of 1 mM (4-aminophenyl)mercuric acetate (APMA) or 0.2 μM trypsin or a solution containing proMMP-7 is heated at 53° C. (cf. for instance, Non-patent reference 7). They revealed that, after a low concentration (less than 1 mg/ml) of activated MMP-7 (also known as Matrilysin) was stored at −20° C. for 6 months and at room temperature for 28 days, there was no change in its activity and behavior on electrophoresis. Although there is no definite description about the change, they appear to suggest that no decomposition of Matrilysin was observed from the results of electrophoresis. In addition to these, there are various reports on purification of proMMP-7 and MMP-7 such as Kihira, and Oneda et al. (cf. for instance, Patent reference 1, Non-patent reference 8) and thus a method for the purification of MMP-7 has been established to some extent at an experimental level. In general, a method for the purification of MMP-7 at an experimental level is scaled up for large-scale production. However, a method for the manufacture of MMP-7 in a large amount has never been established. There is little report on the problems in the manufacture of MMP-7 in a large amount and solution therefor.
For a composition comprising MMP-7, that which comprises tris(hydroxymethyl)aminomethane hydrochloride (Tris hydrochloride), calcium chloride and sodium chloride is reported (cf. for instance, Patent references 3 to 5, Non-patent references 7, 9). It is known that metalloproteinase such as MMP-7, in a state of a solution composition, is stabilized in the coexistence of calcium chloride and sodium chloride (cf. for instance, Patent reference 6). In particular, a medicine is normally required to have an osmotic pressure of around that of body fluid in view of safety. Sodium chloride is commonly used as an osmotic pressure regulator of a liquid composition. As a practical matter, most of the compositions disclosed in these documents comprise a monovalent cation compound such as sodium chloride at the concentration isotonic to or more than that of body fluid. Besides, the compositions disclosed in these documents do not comprise sugar alcohols or sugars.